Citrus Aurantium L.)
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processes Article Yield, Phytochemical Constituents, and Antibacterial Activity of Essential Oils from the Leaves/Twigs, Branches, Branch Wood, and Branch Bark of Sour Orange (Citrus aurantium L.) Mohammad K. Okla 1, Saud A. Alamri 1, Mohamed Z.M. Salem 2,* , Hayssam M. Ali 1,3, Said I. Behiry 4, Ramadan A. Nasser 2, Ibrahim A. Alaraidh 1, Salem M. Al-Ghtani 5 and Walid Soufan 6 1 Botany and Microbiology Department, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia; [email protected] (M.K.O.); [email protected] (S.A.A.); [email protected] (H.M.A.); [email protected] (I.A.A.) 2 Forestry and Wood Technology Department, Faculty of Agriculture (El-Shatby), Alexandria University, Alexandria 21545, Egypt; [email protected] 3 Timber Trees Research Department, Sabahia Horticulture Research Station, Horticulture Research Institute, Agriculture Research Center, Alexandria 21526, Egypt 4 Agricultural Botany Department, Faculty of Agriculture (Saba Basha), Alexandria University, Alexandria 21531, Egypt; [email protected] 5 Biology Department, University College of Taymma, University of Tabuk, Taymma, Tabuk P. O. Box 741, Saudi Arabia; [email protected] 6 Plant Production Department, Faculty of Food & Agriculture Sciences, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia; [email protected] * Correspondence: [email protected]; Tel.: +20-1012456137 Received: 11 May 2019; Accepted: 27 May 2019; Published: 11 June 2019 Abstract: In the present work, essential oils (EOs) extracted from different parts of sour orange Citrus aurantium (green leaves/twigs, small branches, wooden branches, and branch bark) were studied through gas chromatography coupled with mass spectrometry (GC/MS). Furthermore, the EOs in the amounts of 5, 10, 15, 20, and 25 µL were studied for their antibacterial activity against three pathogenic bacteria, Agrobacterium tumefaciens, Dickeya solani, and Erwinia amylovora. The main EO compounds in the leaves/twigs were 4-terpineol (22.59%), D-limonene (16.67%), 4-carvomenthenol (12.84%), and linalool (7.82%). In small green branches, they were D-limonene (71.57%), dodecane (4.80%), oleic acid (2.72%), and trans-palmitoleic acid (2.62%), while in branch bark were D-limonene (54.61%), γ-terpinene (6.68%), dodecane (5.73%), and dimethyl anthranilate (3.13%), and in branch wood were D-limonene (38.13%), dimethyl anthranilate (8.13%), (-)-β-fenchol (6.83%), and dodecane (5.31%). At 25 µL, the EO from branches showed the highest activity against A. tumefaciens (IZ value of 17.66 mm), and leaves/twigs EO against D. solani and E. amylovora had an IZ value of 17.33 mm. It could be concluded for the first time that the wood and branch bark of C. aurantium are a source of phytochemicals, with D-limonene being the predominant compound in the EO, with potential antibacterial activities. The compounds identified in all the studied parts might be appropriate for many applications, such as antimicrobial agents, cosmetics, and pharmaceuticals. Keywords: GC–MS; hydrodistillation; antibacterial activity; clevenger; Citrus aurantium; phytochemical; essential oils Processes 2019, 7, 363; doi:10.3390/pr7060363 www.mdpi.com/journal/processes Processes 2019, 7, 363 2 of 15 1. Introduction Natural extracts and essential oils (EOs) extracted from aromatic and indigenous plants have a broad spectrum of biological activities such as antibacterial, antifungal, antioxidant, anticancer [1–8]. EOs from Citrus spp., especially from peels, have been studied extensively in many research projects over the past few decades [9–11]. They have exhibited bioactivity potentials against the growth of pathogenic bacteria, fungi, and insects [12,13]. The main chemical compounds identified in the EOs from Citrus were limonene, α-pinene, β-pinene, citral, linalool, myrcene, γ-terpinene, eugenol methyl ether, neral, geranial, neryl acetate, and β-caryophyllene [14–18]. The Citrus plants have many biological and aromatic properties because of the occurrence of EOs, alkaloids, glycosides, flavonoids, tannins, and other compounds in its various parts [19,20]. Citrus aurantium L. (Rutaceae), known as sour or bitter orange, is extensively consumed in Mediterranean countries as marmalade and a flavoring agent [21]. The extracted oils have been recognized as safe for their wide uses as antibacterial, antifungal antioxidant, anti-inflammatory, and anxiolytic effects [22–25], and have analgesic activity [26]. Limonene was determined as the main component of bitter orange peel EO, followed by β-myrcene, linalool, β-pinene, and α-pinene [27]. The major compound in Tunisian neroli EO extracted from C. aurantium blossoms is 25.7% linalool [28]. The (R)-(-)-linalool was 59–64% in Citrus (south and south-central Brazil), whereas the hydrolate (orange water) of C. aurantium has nootkatone (17%), α-terpineol (10%), linalool (10%), and limonene (0.8%) [29]. At maturity, limonene exhibited the highest level, with several minor compounds, including linalool, myrcene, and α-terpineol, in the EOs from bitter orange peel [30]. Limonene (92–95%) with linalool and linalyl acetate (together 0.3–3.2%) were identified in the EOs from living (fruits that are still on the tree) bitter orange peel [31]. Shen et al. 32] showed the anti-inflammatory potential of EO from blossoms of C. aurantium L. var. amara Engl with major constituents of linalool, α-terpineol, (R)-limonene, and linalyl acetate [32]. C. aurantium zest EO is composed of limonene (85.22%), β-myrcene, and α-pinene as the main compounds [13]. EO of sweet orange zest consisted of limonene as the main compound, followed by myrcene, α-farnesene, and γ-terpinene [33,34], whereas the EO of sweet orange zest from Uganda and Rwanda contained limonene, myrcene, α-pinene, and linalool [35]. Using the hydrodistillation method, the linalool and terpenes were found to be the main compounds in Neroli blossom EO, whereas, in water recovered oils, linalool, linalyl acetate, geraniol, α-terpineol, and nerol were the main compounds [36]. In flowers, the oil showed the presence of camphor, thymol, linalool, carvacrol, and borneol as main compounds with significant anti-oxidant effect [37]. The goal of the present work was to identify the aromatic chemical profile and antibacterial activity of the EOs from different parts of C. aurantium that could be suitable for different industrial purposes. 2. Materials and Methods 2.1. Plant Material of C. aurantium Fresh branches of C. aurantium were collected in 2019, from Alexandria, Egypt, during pruning process for the trees. The resultant materials were separated to leaves/twigs, small green branches, branch wood, and branch bark. The wood and bark of branches were separated. All the materials were washed with tape water to remove the dust, then cut to small pieces by using scissors to facilitate the extraction process of essential oils (EOs). 2.2. Extraction of EOs Approximately 100 g from each of leaves/twigs, branches, the wood of branches, and branch bark from C. aurantium were soaked in 2 L flasks with 1500 mL of water and hydrodistillated for 3 h in a Clevenger-type apparatus [38]. The distillates of the EOs were dried over anhydrous Na2SO4, filtrated, and measured with respect to the mass of fresh weight of raw material (Table1). The EOs Processes 2019, 7, 363 3 of 15 from leaves/twigs (Petitgrain), branches (2–4 cm in diameter), the wood of branches, and branch bark were kept dry in sealed Eppendorf tubes and stored at 4 ◦C prior to chemical analyses. Table 1. Oil yield from different parts of Citrus aurantium. Oil Yield Part Used (mL/100 g Material) Leaves/twigs 3.45 Branches 1.55 Wood of 1.15 branches Branch bark 1.10 2.3. Gas Chromatography–Mass Spectrometry (GC–MS) Analysis The chemical composition of the essential oils was determined using a Trace GC Ultra-ISQ mass spectrometer (Thermo Scientific, Austin, TX, USA) with a direct capillary column TG–5MS (30 m 0.25 mm 0.25 µm film thickness). Initially, the column oven temperature was held at 45 C, × × ◦ then increased by 5 ◦C/min to 250 ◦C and held for 2 min, then increased to 280 ◦C by 10 ◦C/min. The injector and MS transfer line temperatures were kept at 250 ◦C. Helium was used as a carrier gas at a constant flow rate of 1 mL/min. The solvent delay was 2 min and diluted samples of 1 µL were injected automatically using an Autosampler AS1310 coupled with the GC in the split mode. EI mass spectra were collected at 70 eV ionization voltages over a range of m/z of 40–600 in full scan mode. The ion source was set at 200 ◦C. Identification of the constituents was performed on the basis of their retention times and by comparing the mass spectra with those found in the library search (NIST and Wiley) [39]. Type threshold values contained in Xcalibur 3.0 data system of GC/MS were used as match factors and to confirm that all mass spectra are appended to the library with measuring the Standard Index (SI) and Reverse Standard Index (RSI), where the value 650 is acceptable to confirm ≥ the compounds [40]. 2.4. Antibacterial Activity Antibacterial evaluation of the EOs was assayed against three phytopathogenic bacteria, Agrobacterium tumefaciens, Dickeya solani, and Erwinia amylovora (Microbiology Laboratory, Agricultural Botany Department, Faculty of Agriculture (Saba Basha), Alexandria University, Egypt). The antibacterial evaluation test of the studied four EOs was performed by measuring the inhibition zones (IZs) in millimeters around the loaded filter papers with different amounts of oils (5, 10, 15, 20, and 25 µL) using disc diffusion method [40,41]. Sterile filter paper discs (Whatman filter paper no. 1) with a diameter of 4 mm loaded with different amounts of the studied EOs were placed on the surface of prepared agar plates.